A synthetic biomaterial that mimics the mechanical strength, resorbability, and composition of natural bone hydroxyapatite (HAP) and collagen is not currently available. Synthetic grafts compromising properties between initial strength and resorbability are not ideal for repairing critical sized defects. Thus, large bone defects are not well addressed with current synthetic materials leading to significant impairments of biological function, appearance, and patient quality of life. This project proposes to develop a rigid, bio-inspired material using principles of 1) self-organization of HAP nanocrystals in gelatin molecule and 2) pH-induced calcium- ligand cross-links inspired from the mussel adhesive protein - dopamine. Strong pilot data support all of the proposed aims. Our polydopamine laced HAP-gelatin nanocomposite (PDHG) has compressive and tensile strength approximating 90% and 60% of cortical bone, respectively, and is degradable in vitro. We believe that incorporation of a dopamine-grafted long chain polymer can further improve tensile strength and portends the Long Chain enhanced PDHG (LcPDHG) porous scaffold applicable for CSD repair. The dopamine released from the scaffold also produces positive effects on osteogenesis. The long-term goal of this team is to engineer LcPDHG to fulfill the initial biomechanical requirements and to be eventually resorbed and replaced by endogenous bone. The objective in this particular application is to identify how the incorporation of long chain polymers affects the physical properties (e.g., tensile strength, degradation) of LcPDHG, and how the natural bone and stem cells respond to free dopamine released from LcPDHG. The central hypothesis is that the LcPDHG is a bioactive material with adequate mechanical strength, osteoconductivity and resorption potential to serve as a load bearing graft in CSDs in craniofacial and other skeletal areas. To test this hypothesis three specific aims are proposed: (1) Elucidate the mechanism by which LcPDHG enhances mechanical properties of PDHG to approximate natural bone, and increases its in vitro degradation; (2) Determine cellular mechanisms by which dopamine promotes bone regeneration in the LcPDHG scaffold; and (3) Assess bone formation and replacement of graft materials in LcPDHG scaffolds in a rat calvarial critical sized defect model. Preliminary data predict promising interactions between osteoblasts and dopamine stimuli, and suggest novel signaling via dopamine receptors to promote stem cell-based therapy. The proposed research is significant because it will advance and expand the understanding of how dopamine can be used in bone tissue engineering (TE) and provide the first hydroxyapatite and collagenous artificial bone TE scaffold to repair large bone defects. With a graft material of this type, it should be possible to eliminate multiple surgeries and simplify the treatment of critical-size cranial and facial bone defects.